Human embryonic stem cells (hESCs) are pluripotent cells capable of unlimited self-renewal and can differentiate into the cell types of all three germ layers. The first use of hESCs heralded a new era in regenerative medicine because under appropriate conditions hESCs respond to external signals and can be coaxed to differentiate into specialized cell types such as functional cardiomyocytes and pancreatic β cells. These characteristics make them a valuable cell resource in regenerative medicine. Patients suffering from neurodegenerative, autoimmune, cardiovascular and hematopoietic diseases are potential beneficiaries of stem cell therapy. Despite their tremendous potential, immune rejection of allogeneic hESC-derived cells is a major obstacle to the use of the cells in clinic. Cell surface expression of human leukocyte antigens (HLA), which are encoded by genes in the major histocompatibility complex are the major immunologic barrier.
Unique properties of hESCs can be reestablished in somatic cells by somatic nuclear transfer (SNT) or forced expression of 4 transcription factors Oct4 (O), Sox2 (S), Klf4 (K), and c-Myc (M), which can induce somatic cells into ESC-like cells, which are named induced pluripotent stem cells (iPSCs). These two approaches allow derivation of patient specific pluripotent stem cells. Importantly, since these cells are made from a patient's own cells, it is considered that their immune system will not reject them.
However, since SNT mediated reprogramming of human somatic cells is in its infancy, has low efficiency and requires oocyte donation, it cannot yet offer a practical solution. While relatively simple derivation of iPSCs seems promising, according to recent reports, transcription factor-based reprogramming is associated with incomplete epigenetic reprogramming. Therefore using these cells in clinic requires detailed examination of iPSC clones, which is cumbersome. Moreover, generating pluripotent stem cells under good manufacturing practices (GMP) for individual patients is likely to be financially prohibitive.
One approach to overcoming the immunological barrier to stem-cell transplantation is to establish clinical-grade hESC/iPSC/SNT-hESC banks with HLA haplotypes, which will match a significant proportion of the population. However derivation of hESC/iPSC/SNT-hESC lines under current good manufacturing practice (cGMP) requires investments of substantial amounts of money and time.
Another solution to avoid immune rejection of hESC derivatives is genetic manipulation of HLA molecules. By using zinc finger nucleases, Torikai and colleagues selectively eliminated human leukocyte antigen (HLA) class I in ESCs and demonstrated that HLA-A cells could escape lysis from HLA-restricted cytotoxic T lymphocytes. However HLA class I complete knock-out cells are targets for NK-cell-mediated cytotoxicity (Oberg L etc., Eur J Immunol., 2004, 34(6): 1646-1653).
In another study, Riolobos and colleagues disrupted beta-2 microglobulin (B2M) which encodes the accessory chain of major histocompatibility complex (MHC) class I molecules and is required for their surface expression (Laura Riolobos etc., 2013, 21(6): 1232-1241). Therefore, the homozygous deletion of the B2M gene prevents the surface translocation of class I HLA molecules and reduces immunogenicity. However this approach offers a limited solution because it has been reported that hematopoietic stem cells lacking the B2M gene are eliminated by NK cells. Although enforced expression of less polymorphic HLA-E or HLA-G-B2M chimeric proteins protects class I negative cells from NK-cell-mediated lysis in vitro, any in vivo data has not been reported yet.
More recently, cytotoxic T lymphocyte-associated protein 4 (CTLA4)-immunoglobulin and programmed cell death ligand 1 (PDL1) knock-in human ESCs (hESCs) were shown to simultaneously disrupt T cell costimulatory pathways and activate T cell inhibitory pathways in humanized mice. It is well known that infected cells are also subject to immune response. There is no in vivo study showing if these CTLA4 and PDL1 double knock-in cells can be eliminated by immune cells when they become infected.
It appears the monoallelic mutation of HLA molecules and consequent derivation of HLA homozygous hESCs might offer a solution for overcoming immune rejection of hESCs. Creation of a small library of homozygous hESCs from existing hESC lines could cover significant percentage of the human population.